There are increased market pressures on the healthcare industry to provide protein based therapeutics in ample amounts while being “specific pathogen free” (“SPF”) and at reduced cost. Clinical validation is still an overriding cost to process restructuring as well as the introduction of new protein based drugs. Needs for increases in process efficiency include that of pre-purification steps and affinity adsorption processes which can select for desirable protein populations. Affinity and ion exchange adsorption technology are scaleable technologies important to protein purification. Ideally, a new adsorption technology will have high selectivity, yield, throughput, and will be amenable to or directly provide improved specific pathogen removal, minimized buffer usage, minimized operating complexity, and also be less expensive to produce than current matrices. Therefore, specific advantages imparted by new matrix technologies should be amenable to process validation.
I. Inorganic Oxide-Based Chromatographic Supports
Currently known inorganic chromatography supports comprising particulate silica (SiO.sub.2) or alumina (Al.sub.2 O.sub.3) are stable over pH ranges of about 1-8 and 3-12, respectively. The solubilization of SiO.sub.2 and Al.sub.2 O.sub.3 at pHs outside of these ranges results in deterioration of these supports and contamination of the resultant chromatographed and separated products with silicon- or aluminum-containing species. Methods of improving the alkaline stability of particulate SiO.sub.2 by cladding the surface with a more base stable metal oxide such as zirconium oxide (ZrO.sub.2) have been disclosed in U.S. Pat. Nos. 4,648,975 and 4,600,646. This cladding is disclosed to increase the upper pH limit at which these supports, also referred to as packings, can be used to 11 and 9.5, respectively. However, these packings still lack adequate stability to allow them to be sterilized and cleaned in, for example, 0.1N aqueous sodium hydroxide (NaOH, pH=13).
Use of porous spherical ZrO.sub.2 particles on a thin layer chromatography plate has been disclosed in U.S. Pat. No. 4,138,336, a process for the preparation of porous ZrO.sub.2 microspheres is taught in U.S. Pat. No. 4,010,242, and chromatographic use of these particles is taught in U.S. Pat. No. 3,782,075. The microspheres are prepared by a process in which colloidal metal oxide particles are mixed with a polymerizable organic material and coacervated into spherical particles by initiating polymerization of the organic material. This is a time consuming, batch process which requires the addition of organic material which is pyrolized and hence lost.
U.S. Pat. No. 3,862,908 discloses microspheres of urania and other metal oxides; however, these particles are fired to near full density, have reduced surface areas and therefore, would not be attractive for chromatographic uses.
U.S. Pat. No. 3,892,580 discloses a process for preparing porous bodies of ZrO.sub.2. This process requires the use of a binder to react with the oxide particles during preparation. This binder is subsequently decomposed by pyrolysis and therefore lost. The bodies produced by this process are not spherical, would pack unevenly, may cause increased column pressure, and are therefore not attractive for chromatographic uses.
U.S. Pat. No. 4,389,385 teaches the preparation of porous gels and ceramic materials by dispersing solid particles of an inorganic substance produced by a vapor phase condensation method in a liquid to form a sol. The sol contains colloidal particles which are aggregates of the primary particles. The sol is dried to produce a porous gel of greater than 70% by volume porosity.
The eluent, also referred to as the mobile phase, used to elute the various components from the stationary phase is relatively polar, e.g., an aqueous buffer or a mixture of water and an organic solvent, e.g., aqueous alcohol. Its polarity can be changed by increasing the concentration of the less polar liquid in the mobile phase, a technique known in the art.
Thus relative to the use of ZrO.sub.2-clad silica, a more promising approach to developing a highly stable reversed-phase support, involves replacing the silica with an alternative inorganic material, such as alumina. Although it has been demonstrated that some improvement in pH stability is realized by replacing silica with alumina, the dissolution of alumina in aqueous solutions at extreme pHs (pH<2 and pH>12), even at room temperature, is well known.
As mentioned previously, in addition to the use of a pH-stable support material, the production of a stable, reversed-phase also requires a process for modifying the support material which results in a stable, hydrophobic surface. Silylation is the most widely used method to derivatize silica particles to produce hydrophobic reversed-phase supports. The silylation of inorganic bodies other than silica (e.g., alumina, titania, zirconia, etc.) has been disclosed in U.S. Pat. No. 3,956,179. However, it is uncertain whether or not covalent bonds to the support surface are actually formed. In any event, the hydrolytic instability of the siloxane bond is well known, and it is very likely that a Si—O-metal bond will be even more susceptible to aqueous hydrolysis because of the increased polarity of the bond.
An alternate approach to silylation for modifying the surface polarity of inorganic bodies is the sorption of a polymer of desired polarity/functionality onto an SiO.sub.2 or Al.sub.2 O.sub.3 support surface followed by cross-linking of the individual polymer chains to one another to impart additional stability to the coating. Reversed-phase supports prepared in this fashion exhibit much improved pH stability compared to those prepared by silylation. It is important to recognize that the formation of a stable, cross-linked polymer layer on the surface of the support does not reduce the need for a stable, inorganic support, since it may not be possible to cover the entire inorganic surface. Although cross-linking of the polymer may keep it in place even as the underlying inorganic support dissolves, dissolution of the support will undoubtedly lead to a reduction in the mechanical stability of the support. In addition, problems related to increasing column back pressure are known to accompany the dissolution of the inorganic support and its subsequent appearance in the mobile phase and transport through the column and the accompanying instrumentation.
Another problem related to the use of silica-based reversed phase supports is the difficulty encountered in the chromatography of amines and other basic solutes. This problem results from the presence of acidic silanol groups (SiOH) on the silica surface. Basic solutes undergo very strong interactions with these silanol groups which may involve cation exchange or hydrogen bonding, depending on the pH of the mobile phase. This problem is exaggerated by the requirement of working in the pH range 2<pH<8 on silica-based columns, since most amines will be protonated in this pH range and protonated amines can readily bond to the silica surface. One obvious approach to improving the chromatography of amines is to work at hydrogen ion concentrations significantly lower than the ionization constant of the amines so that they are unprotonated. For aliphatic amines, this normally involves working at a pH greater than 11. However, these pH ranges cannot be employed using silica-based columns.
The presence of the aforementioned acidic silanol groups can also lead to irreversible adsorption of many classes of organic molecules onto silica-based reversed-phase supports, a problem which is well known to those versed in the art. This irreversible adsorption is can be particularly troublesome in the reversed-phase HPLC of proteins. Ultimately, this adsorption will result in a change in the properties of the support and can lead to its destruction. Thus, alternative materials are desired for chromatographic separations.
II. Ion-Exchange High Pressure Liquid Chromatography
Ion-exchange chromatography (IEC) has become an important separation technique for the purification of biomolecules. Typical supports used in IEC are silica, alumina, agarose, polymethacrylate, and poly(styrenedivinylbenzene). See H. G. Barth et al., Anal. Chem., 60, 387R (1988). Agarose is not suitable for high pressure work, while silica and alumina have limited pH stability. The matrices of silica and alumina must also be derivatized or coated to provide the support with ion exchange properties. This often introduces hydrophobic interactions into the retention mechanism. The hydrophobic nature of hydrocarbon-based supports such as poly(styrene-divinylbenzene) must be masked in order for them to be used as IEC supports. The hydrocarbon-based supports are also subject to shrinking and swelling whereas inorganic supports are not.
Zirconium phosphate has been extensively studied as an inorganic ion exchanger for the nuclear industry because of its excellent exchange capacities, radiation and thermal stability. See A. Clearfield et al., Ion Exchange and Solvent Extraction, J. A. Marinsky et al., eds., Marcel Decker, New York, (1973) at Chapter 1.
Ion-exchange chromatography is the leading technique in the purification of proteins [Sofer, G., J. Chromatogr., 707 (1995), 23.]. Mobile phase conditions used in the ion-exchange mode are generally non-denaturing leading to high recoveries and retention of biological activity encountered when purifying bio-polymers by this method [Kopaciewicz, W., Rounds, M. A., Fausnaugh, J. and Regnier, F. E., J. Chromatogr., 266 (1983), 3.]. In addition, elution profiles are relatively predictable facilitating scale up [Yang, Y.-B. and Regnier, F. E., J. Chromatogr., 544 (1991), 233.].
The popularity of ion-exchange chromatography for protein purification has grown with advances in ion-exchange stationary phase supports [Yang, Y.-B., Harrison, K. and Kindsvater, J., J. Chromatogr., 723 (1996), 1.]. This progression towards better ion-exchange materials, and column packings in general, includes changes in the base support material. Agarose and silica based phases are being used less in favor of highly crosslinked polystyrene divinylbenzene (PS-DVB) [Sofer, G., J. Chromatogr., 707 (1995), 23., 8-11; Lee, D. P., J. Chromatogr. Sci ., 20 (1982), 203.; Dawkins, J. V., Lloyd, L. L. and Warner, F. P., J. Chromatogr., 352 (1986), 157.; Bowers, L. D. and Pedigo, S., J. Chromatogr., 371 (1986), 243.; L. Varaday, N. Mu, Y.-B. Yang, S. E. Cook, N. Afeyan and F. E. Regnier, J. Chromatogr., 631 (1993) 107.] as well as metal oxide supports such as alumina, titania [K. K. Unger, in P. R. Brown and R. A. Hartwick (Editors), High Performance Liquid Chromatography, Wiley, New York, 1989, Ch. 3, p. 145.] and zirconia [M. Kawahara, H. Nakamura and T. Nakajima, Anal. Sci., 4 (1988), 671.; M. Kawahara, H. Nakamura and T. Nakajima, Anal. Sci., 5 (1989), 485.; M. Kawahara, H. Nakamura and T. Nakajima, J. Chromatogr., 515 (1990), 149.; U. Trüdinger, G. Müller and K. K. Unger, J. Chromatogr., 535 (1990), 111.; J. Nawrocki, M. P. Rigney, A. V. McCormick and P. W. Carr, J. Chromatogr., 657 (1993), 229.] owing to the increased pH and thermal resistance of newer supports towards dissolution, and their ability to accommodate organic solvents without shrinking or swelling [C. B. Amphlett, L. A. McDonald and M. J. Redman, J., Inorg. Nucl. Chem., 6 (1958), 236.; C. B. Amphlett, L. A. McDonald and M. J. Redman, J., Inorg. Nucl. Chem., 6 (1958), 220.; N. Michael, W. D. Fletcher, D. E. Croucher and M. J. Bell, Report CVNA-135, Carolina-Virginia Nucl. Power Assoc., Charlotte, N.C., 1961.].
A widely used method for the preparative purification of antibodies [Malm, B., “A Method Suitable for the Isolation of Monoclonal Antibodies from Large Volumes of Serum-Containing Hybridoma Cell Culture Supernatants,” J. Immunological Methods, 104 (1987), 103-109.] involves the use of three LC columns, first a cation-exchange column, followed by an anion-exchange column and finally an affinity column as the final purification media. This method is depicted in FIG. 1. This purification protocol is extremely time-consuming, but the final product is adequately pure (>95%). The first two columns in the purification protocol are an anion-exchange and a cation-exchange column. These two columns are used as initial clean-up columns and are followed by an affinity column that specifically binds and then releases the pure monoclonal antibody.
A single EDTPA modified metal oxide column offers a cost-effective alternative to this three column approach. Furthermore, the extreme stability of zirconia stationary phases makes the media sterilizable and cleanable thereby further reducing antibody production costs. The main benefits, among others, of metal oxide-based Mab purification media are:                1. New Cost-Effective Alternative to Protein A and Protein G Affinity Chromatographic Media;        2. Particles are Made from Inert Zirconium Dioxide with A Non-Animal Source Stationary Phase;        3. Particles are Chemically Stable in Acid and Base Solutions, which Allows for Depyrogenation and Cleaning of Particles;        4. Rigid Particles Allow for Use of High Linear Velocities for Unparalleled Product Throughput;        5. Useful for a Variety of Immunoglobulins Including IgGs, IgA and IgMs;        6. Does Not Bind Serum Proteins that can “Foul” Protein A and Protein G Media;        7. Tunable Selectivity for Different Immunoglobulins Using pH and Ionic Strength as the Main Variables;        8. Antibody Purity Levels As Good or Better than Protein A and Protein G Media;        9. Extended Media Lifetime Compared to Protein A and Protein G Media;        10. High Binding Capacity for Immunoglobulins to Protein A and Protein G Media.        